PROJECT SUMMARY Liver transplantation, currently the only treatment for end-stage liver disease (ESLD), is limited by a severe shortage of donor organs. Cell- and tissue-based approaches have thus far failed to produce viable alternative therapies to meet this critical clinical need. The goal of the proposed research is to develop an entirely new strategy that embraces the principles of regenerative medicine to surgically replace non-functional liver tissue. It is based on the concept that a supportive niche must first be created in the liver in order to facilitate engraftment of new cells. Non-thermal irreversible electroporation (NTIRE) is a tissue ablation technology that causes cell death while preserving the extracellular matrix and inducing minimal inflammation. While NTIRE is currently used clinically as a tumor ablation modality, we propose a novel application for NTIRE as the key technology for producing a decellularized tissue scaffold in vivo and in situ that is a supportive niche for new cell engraftment. We recently showed that exogenous hepatocytes successfully engrafted into host liver parenchyma that was pre-treated with NTIRE, demonstrating the feasibility of our proposed approach. Further development of this new application requires optimizing NTIRE electrical parameters to induce non- inflammatory cell death and clearance of dead cells by resident phagocytes, leading to pristine preservation of the niche that promotes new cell growth. These conditions were not important for the purposes of tumor ablation and have not been defined. In addition, most ESLD occurs in the setting of liver fibrosis and the effect of NTIRE on fibrotic tissue is unknown. Our preliminary studies suggest that a fibrotic liver responds to the same applied electrical field differently than a normal non-fibrotic liver. There is also ongoing inflammation and presence of profibrogenic macrophages in a fibrotic liver that may change the nature of the immune response typically induced by NTIRE treatment of non-fibrotic liver. We hypothesize that the NTIRE electrical parameters that induce non-inflammatory cell death and subsequent clearance by phagocytes are different in normal and fibrotic liver. Therefore, to understand how to optimize conditions for successful exogenous cell engraftment to treat ESLD, we will perform a titration of NTIRE electrical parameters and investigate the corresponding mechanisms of NTIRE-induced cell death (Aim 1) and its modulation of the immune response (Aim 2) in both normal non-fibrotic liver and diseased fibrotic liver. These basic discoveries of NTIRE's mechanism of action can then be utilized to develop the most effective strategies for introducing exogenous cells into a host liver in vivo for tissue replacement therapy. The proposed research is the exploratory first step toward developing a novel surgical procedure that would be a form of ?minimally-invasive liver transplantation.? If successful, it would revolutionize the treatment of ESLD and pioneer a field of regenerative surgery.